Advances in Bioscience and Biotechnology, 2013, 4, 8-14 ABB
http://dx.doi.org/10.4236/abb.2013.410A4002 Published Online October 2013 (http://www.scirp.org/journal/abb/)
Utility of adeno-associated viruses to target members of the
TGF-β superfamily in prostate cancer therapy
Priya Ramarao, Elspeth Gold
Department of Anatomy, University of Otago, Dunedin, New Zealand
Received 28 July 2013; revised 28 August 2013; accepted 15 September 2013
Copyright © 2013 Priya Ramarao, Elspeth Gold. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Components of the TGF-β superfamily have been
well established in their intricate and multifaceted
roles in canc er prog ress ion and surv ival . The TGF- βs
have been targeted therapeutically in an attempt to
modify complex tumour networks to favour cancer
cell destruction. Goals of these therapies are often to
attack the “hallmarks” of cancer: characteristics ac-
quired by cancer cells via re-wiring or manipulating
existing biological pathways to their survival advan-
tage. Of the multitude of targeted therapies currently
available, viral therapies have shown much promise
in their efficacy of treatment. This review highlights
current viral therapies targeting members of the
TGF-β superfamily, with a focus on the strengths
and limitations associated with this form of targeted
cancer therapy.
Keywords: AAV; Prostate Cancer; TGF-β Superfamily
1. PROSTATE CANCER
One of the leading causes of cancer related deaths in de-
veloped countries, prostate cancer, affects a large number
of men. The progression of the cancer from its initiation
in the prostate, which often precedes dispersion as me-
tastases to tissues such as bone, lymph, liver and brain
leaves several window s of opportunity during which cer-
tain therapies can be administered. While several treat-
ment methods hav e been developed targeting a multitude
of aspects of tumour characteristics, acquisition of resis-
tance is a familiar roadblock encountered by investigators,
and circumventing this obstacle remains a difficult task
[1].
2. ROLES OF THE TGF-βS
Despite partaking in a variety of developmental process-
es crucial for normal development, aberrant reactivation
of these TGF-βs often results in tumorigenic behaviour
[2]. The roles they partake in during tumourigenesis of-
ten reflect their roles in embryonic development, but also
extends to other features often observed in cancer, such
as cachexia and bone loss [3]. A re-deployment of their de-
velopmental roles to the advantage of the tumour is ob-
served. At the stages of prostate tumourigenesis, increas-
ed TGF-β production results in extracellular matrix deg-
radation, immunosuppression and angiogenesis, all resul-
ting in evasion of cell death and increased cell survival,
favouring cancer cell propagation [4]. The conundrum
faced when targeting TGF-β is the fact that in the earlier
stages of prostate tumourigenesis, TGF-β exerts growth
inhibitory effects on cells, which is a desirable character-
istic in cancer treatment, whereas in later stages encour-
ages tumour growth [4]. As such, understanding the un-
derlying rewiring of pathways that occur before this fun-
damental switch are essential to capitalise favourable tu-
mour suppressive roles of TGF-β, while simultaneously
minimising its tumour promot ing rol e.
The TGF-β Superfamily
Members of the TGF-β superfamily consist of TGF-βs,
BMPs, NODAL, activins and GDFs. NODAL influence
the tumour parenchyma, whereas activins and GDFs have
been found to modulate the tumour microenvironment.
The core of the TGF-β signalling cascade is shared and
similar across all TGF-β superfamily members. Ligands
of this superfamily dimerise either as homodimers or he-
terodimers, bridged by a disulphide bond. Receptors for
these homo/hetero-dimers are a complex of two receptors,
Type 1 and Type II, which also associate with each other
to form a double complex. In its ligand bound state, the
Type II receptor phosphorylates its partnered Type I re-
ceptor, thereby activating the kinase domain of the Type
1 receptor. As a result, downstream effectors such as

P. Ramarao, E. Gold / Advances in Bioscience and Biotechnology 4 (2013) 8-14 9
SMADs are phosphorylated by the Type I receptor
(SMAD2/3 for activins, TGF-βs, GDFs and NODAL,
SMAD1/5/8 for BMPs), and this conformational change
allows the association with SMAD4/5. Nuclear import is
made permissible by this association, and the complex is
able to bind a variety of other proteins to fine-tune tran-
scription (Figure 1).
SMADs recruit chromatin remodellers which are able
to either activate or repress gen e transcription, depending
on whether they promote chromatin condensation or
chromatin expansion. One of the many layers of regula-
tion of SMAD activity include the upstream antagonists
of the ligands. SMADs are phosphorylated at several dif-
ferent sites to modulate their activity; these modifications
are catalysed by mediators of other signalling pathways
such as the MAPK pathways [5-7]. NODAL is required
for epithelial-to-mesenchymal transition as well as cell
sorting. Intriguingly, the signalling strength is able to
dictate the effect on surrounding cells. It has also been
found that high levels of PI3K activity induce pluripo-
tency, whereas low levels induce mesenchymal differen-
tiation. These relate to Wnt and ERK signalling to pat-
tern the anterio-posterior axis as well as the dorso -ventral
axis. BMPs and GDFs are involved in maintaining and
overseeing adult tissues, whereas NODAL is expressed
embryonically; adult exp ression is often abnormal [8,9].
The role of NODAL goes back to the very early stages
of embryonic development, where it is involved in main-
tenance of pluripotency in the blastocyst. As a morpho-
gen, it forms the basis of the proximal-distal axis, an d in
later stages, the anterior-posterior axis. Its role in pluri-
potency maintenance is also in conjunction with its abil-
ity to influence epithelial-to-mesenchymal transition: a
key requirement for cells during development, to main-
tain the plasticity and alter the d ifferentiation capabilities
of the cells [10]. This plasticity, which comes hand in
hand with increased proliferative abilities and regenera-
tion is re-deployed during tumorigenesis. In fact, normal
adult cells do not express NODAL; ab errant activation in
adult cells are only observed in pathological conditions
[11]. An example of this is in certain prostate cancers,
elevated levels of NODAL are correlated with increased
aggressiveness of the tumour, as well as increased poten-
tial for metastatic dissemination [12].
BMPs often oppose inducers of epithelial to mesenchy-
mal transition. However, a re-wiring of BMP pathways
see an overexpression of BMPs at the sites of metastasis.
Intriguingly, in prostate cancer cells, BMP7 was able to
maintain dormancy of a bone metastasis of prostate can-
cer. This effect was not applicable to the primary tumour,
which shows that interaction with the tumour microen-
vironment may underpin this discrepancy [13].
Figure 1. Signaling cascades of the TGF-β superfamily—Each ligand binds their respective Type II receptor. Upon association of the
ligand with its Type II receptor, phosphorylation of a Type I receptor is catalysed by the Type II receptor. Subsequently, the pho-
sphorylated serine/threonine kinase domain of the Type II receptor in turn phosphorylates Smad 2 or Smad 3 (for activins, TGF-βs,
NODAL and GDF) or Smad1/5/8 for the BMP signaling cascade. Smad complexes which are formed are then able to bind Smad4,
llowing for nuclear transport and transcription of relevant genes. a

P. Ramarao, E. Gold / Advances in Bioscience and Biotechnology 4 (2013) 8-14 10
Activins comprise two inhibin-β subunits of varying
subtypes; βA, βB, βC and βE. The former two subunits
are present in a diverse array of different tissues, whereas
the latter two are found principally in the liver. Antago-
nists of these ligands are composed of an alpha subunit
linked to a beta subunit via a single disulphide bond [14].
Redundancies exist between myostatin , activin and other
TGF-β superfamily members in terms of the consequence
of their interactions with the activin receptors. Activins
have also been discovered to be potent immunosuppres-
sive agents, aiding tumours in immune evasion, another
hallmark of cancer [15]. Increased activin levels, causa-
tive of a lowered inhibin antagonist level causes cancer
cachexia, which is a wasting syndrome observed in many
cancers. Activins and BMPs also play a role in balancing
osleoclastic and osteoblastic activity, which are often
aberrantly activated in instances of bone metastases [16].
Evidently, all the members of the TGF-β superfamily
are able to work synergistically to mold a favourable en-
vironment for tumorigenesis, by sculpting the tumour
microenvironment. While their normal roles are crucial
for development and regular homeostatic processes, ab-
errant reactivation or redeployment can result in highly
unfavourable circumstances which promote tumour pro-
pagation and cancer progression. It is clear that the abil-
ity to target these components in a temporally controlled
way can be of large benefit in cancer therapy to help re-
store the balance in the signalling pathways.
3. POTENTIAL FOR THERAPEUTIC
TARGETING OF TGF-β
Dichotomous roles underpin TGF-β signaling in relation
to cancer progression; in early stages of tumorigenesis,
TGF-β signaling is endowed with the role of a tumour
suppressor, whereas later stages see the signaling path-
way involved in a more tumour promoting role. This mul-
tifaceted relationship poses as an obstacle to successful
cancer therapies. Generally, therapies targeting the path-
way can be directed at three points; at the ligand level,
altering the dynamics of ligand-receptor interactions or
modifying their second messengers.
4. THE ROLE OF TGF-βS IN PROSTATE
CANCER
The TGF-β superfamily (made up of activins, TGF-β,
BMPs, Nodal and GDFs) are involved in an intricate re-
lationship in tumorigenesis, and teasing out the specific
roles of this superfamily during cancer progression has
yielded a wealth of information about their roles in can-
cer progression. In normal cells, TGF-βs are growth in-
hibitors; they promote cell cycle arrest, a necessary step
for growth control and apoptosis. It has been postulated
that at this stage, signaling is carried out through a Smad-
dependent pathway, whereas metastatic dissemination is
attributable to a non-Smad dependent pathway [7]. Addi-
tionally, there is a collaborative interaction between the
androgen receptor and TGF-β1, as the promoter region of
TGF-β1 contains androgen response elements, illustrating
a synergistic control over growth processes. Insensitivity
to the pro-apoptotic signals conferred by the TGF-β sig-
naling pathway is observed in instances whereby andro-
gen receptor (AR) is overexpressed; even in hormone-in-
dependent prostate cancer, AR overexpression constitutes
a dampened sensitivity to growth inhibitory signals elic-
ited by TGF-β pathways [17]. Induction of apoptosis is
observed when TGF-β1 is administered into the pros tate,
as well as an alteration from an epithelial to a mesenchy-
mal nature (EMT), a hallmark of oncogenic transforma-
tion. Interestingly, the aforementioned transformation
precedes dissemination of bone metastases [4].
Much research has been invested into deciphering the
cues and components which facilitate the switching of
TGF-β from tumour suppressor to tumour promoter. In-
triguingly, attenuation of the default TGF-β pathway, with
simultaneous reactivation and rewiring of TGF-β control-
led pathways is often observed in prostate cancer—mani-
festing as increased levels of TGF-β ligands. As such,
there is a clear fundamental connection between TGF-β
and different levels of cancer progression. This begs the
question—what approaches can be used to target these
components?
5. TARGETING TGF-βS WITH VIRUSES
While antibody therapy [18] and antisense oligonucleo-
tides [19] are commonly used therapies to target TGF-βs,
another method with superior targeting accuracy appears
to be viral vectors [20]. The next section aims to discuss
studies which have employed adenoviral-related approa-
ches to target members of the TGF-β superfamily, and
the potential that these approaches may have when tar-
geted to the prostate. Two main methods exist for viral
targeting of cells; oncolytic viruses—which catalyse ubi-
quitous infection but rely on cancer cell-specific replica-
tion machinery, and are promoter driven. The former is
achieved by selecting a promoter which is recognised by
transcription factors and messengers only found in the
cell type which is being targeted, and the latter by alter-
ing the tropism of the virus to target certain receptors
found on the target cell type. In prostate cancer, PSA is
an often-used promoter to drive viral integration or gene
transcription. However, its dependency on androgens,
owed to an AR-dependent enhancer poses as an obstacle
when it is being used alongside androgen deprivation
therapy [21].

P. Ramarao, E. Gold / Advances in Bioscience and Biotechnology 4 (2013) 8-14 11
6. GENE THERAPY WITH VIRAL
VECTORS
6.1. Adenoviruses
Adenoviral vectors require target cells to express a CAR
receptor for viral integration; CAR receptors are often
overexpressed in prostate cancers. Despite this, different
classes of prostate cancer show varying vulnerability to
adenoviral infection. Add ition of a cell-type specific mo-
lecule, such as an antibod y or peptid ecan de liver the v iral
content in a cell-targeted manner. A variety of specific
motifs bind the CAR receptors; upon successful binding,
the virus is assimilated into the cell, where it can carry
out its oncolytic activity.While the main aim for viral de-
livery is to successfully infect target cells, it is equally
vital to “detarget” the virus to ensure minimal delivery to
normal, non-target cells [22]. As such, a wealth of re-
search has been undertaken to pinpoint specific motifs
which will catalyse infection in desired cells o nly. To this
end, directed evolution of viruses is conducted to produce
an array of serotypes able to target different tissues. The
malleability of adenoviral vector fibre knobs make it cu-
stomisable to be used for many tissues.
Two main methods exist for targeting viruses to pre-
ferred cells—transductional and tran scriptional targeting.
The former involves directing the virus to infect a limited
set of cells, preferably cancer cells specifically. The latter
results in ubiquitous infection , without d ifferentiating be-
tween cancer cells and normal cells. Transcriptional tar-
geting can be achieved in two ways. The first method uti-
lises a virus with deleted genes required for viral replica-
tion, only to be compensated for in tumours cells. As such,
the destructive effects are only “activated” in the tumour
cells. Normal cells remain unharmed as they are unable
to compensate for the missing genes. Secondly, the E1A
gene, required for viral replication can be knocked out in
the viral genome, but can be induced in the presence of a
tumour cell-specific promoter. Adenoviral vectors can also
be oncolytic: the mere act of adenoviral replication in
target cells is sufficient to elicit “oncolysis”—cancer cell
death [23].
6.2. Targeting Bone Metastases in Prostate
Cancer
TGF-βs are involved in osteoclastic differentiation and
dissemination of bone metastases. Later stages of cancer
progression result in these bone metastases, with few the-
rapies available to successfully treat this phase of the
disease. Hu et al. [24], developed oncolytic adenoviruses
which simultaneously targeted TGF-β via a protein.
Downregulation of second messenger SMADs was ob-
served upon infection of these cells. Authors postulated
that while the targeted infectivity of the adenoviruses to
cancer cells specifically was sufficient for oncolytic ac-
tivity, antitumour activity was further enhanced by the
effect of SMAD downregulation on the tumour microen-
vironment. Dispersion of signals initiated b y overexpres-
sion of TGF-β into the surrounding support environment
was therefore successfully inhibited, dampening the growth-
promoting effects of the microevironment. Consequently,
the study solidified the long standing reputation of ade-
noviral vectors as a suitable source of tumour targeting.
This is an intriguing form of “armed” oncolytic adenovi-
ruses; “armed” because in addition to oncolytic capabili-
ties of the virus, it harbours a further capacity to down-
regulate second messengers.
6.3. Targeting Cancer Cells with RNA
Interference
Downregulation of components via RNA interference can
be achieved by exploiting natural cellular responses to
double stranded RNA (dsRNA) species—human cells al-
ways consider dsRNA to be foreign, as they are not nor-
mally present in the body. In a recent paper by Oh et al.
[25], adenoviruses expressing TGFβ-1 short hairpin
RNA(shRNA) were introduced into a metastatic prostate
cancer cell line, DU-145, achieving a 98% downregula-
tion of TGFβ-1 mRNA, with a concurrent decrease in cor-
responding protein levels. In contrast with the approach
outlined in the previou s section, which targeted the TGF-
β signaling cascade at the receptor level, this approach
targets TGF-β at the ligand level. Intriguingly, this study
showed that when TGF-β1 was downregulated, a com-
pensatory mechanism is elicited whereby TGF-β3 is up-
regulated. Due to th e fact that TGF-β1 and TGF-β3 have
overlapping functions, this has profound implications for
future therapies targeting the TGF-β ligands, as compen-
satory mechanisms may hinder any anti-tumour therap ies
targeting TGF-β ligands. Another very relevant finding in
this study was the downregulation of both TGF-β1 and
TGF-β3 alongside shRNA mediated degradation of TGF-
β2 ligand. As such, in th e interest of successful therapies
targeting this family of ligands, TGF-β2 may be a more
successful target to ensure faithful downregulation of all
ligands that may have the same function.
6.4. Utilising Immunological Effects to Elicit
Anti-Tumour Responses
A few studies have attempted to take advantage of the
immunological response elicited by adenoviruses to pro-
voke cancer cell destruction. The induction of active im-
munity is highlighted in two prominent studies. Firstly,
inducing oncolysis by means of a suicide gene, Ad5-CD/
TK-rep conjures an immunological response which tar-
gets cells infected by the virus, thus selectively destroy-
ing cancer cells [26]. Another promising study [27] pro-
vokes T-cells to set up immunological responses to an

P. Ramarao, E. Gold / Advances in Bioscience and Biotechnology 4 (2013) 8-14 12
adenovirus containing the PSA promoter, in hopes that
the reaction set up by this process will destroy all PSA
expressing cells. In terms of what this means for TGF-β
targeting, the above strategies can be conducted along-
side a TGF-β knockdown (at either the receptor, ligand
or second messenger level) to produce a powerful antitu-
mour response. Furthermore, background active immuni-
ty may also offer protection from potential future devel-
opment of prostate cancer.
6.5. Specific Targeting to Newcastle Disease
Virus
Another fascinating discovery was the use of a Newcas-
tle Disease Virus (NDV) to specifically target prostate
cancer cells. Incorporation of the virus was dependent on
presence of a substrate (HSSKLQ), which is PSA spe-
cific. Its oncolytic activity was observed even in hormone
refractory prostate cancer [28].
7. ADENO-ASSOCIATED VIRUSES
The very ability of adenoviruses to induce powerful im-
munological responses may also serve to be their down-
fall; with these responses also po tentially preventing sur-
vival of the adenovirus for a duration long enough to car-
ry out infection and subsequent tumour destruction. While
adenoviral virus-based treatments have dominated the
field of gene therapy, Adeno-Associated Virus (AAVs)
are a much more attractive means of achieving tumour
regression, notwithstanding a few drawbacks. They be-
long to a class of dependovirus, requiring the replication
capability of a “helper” virus, such as adenovirus. In the
absence of a helper virus, they succumb to a lysogenic
cycle; they predictably incorporate at a locus on chromo-
some 19 (19q13.3), and then replicate alongside the host
cell genome. When a helper virus is present, the AAV en-
ters a lytic cycle, resulting in rapid replication and dis-
persion of progeny to surrounding cells.
Utility of AAVs in the clinic come with significant
advantages.Weak immune responses are elicited by AAVs,
making it an attractive candidate for gene therapy. Pow-
erful immune responses such as those that can be elicited
by adenoviruses can be co unterpr odu ctiv e as we ll as dan-
gerous to the host. In addition, inflammatory responses
mediated by AAVs are far weaker than those induced by
adenoviruses. The predictable nature of its integration
locus avoids any concern for random insertion and mu-
tagenesis. Despite these advantages, certain features of
AAVs pose as obstacles to successful clinical utility. AAVs
have a much lower infection efficiency compared to ade-
noviruses; 40% vs almost 100%. Its packaging capacity
is also half of that of adenoviruses. Also, because AAVs
only package single stranded DNA, second strand syn-
thesis is required after infection, although this has now
been circumvented by self-complementary Adeno-Asso-
ciated Viruses (scAAVs). Lastly, much like adenovirus es,
peptides embedded in the viral coat of AAVs can be al-
tered to direct them into a wide variety of tissue types,
such as prostate. The variety of prostate-specific genes
and antigens make it an ideal tissue for specific viral tar-
geting, although there is of ten no differentiation between
prostate cancer cells and normal pro state cells. However,
as prostate cancer most often affects the post-reproduc-
tive population, th is is not a huge obstacle. The following
section aims to highlight some studies which have tar-
geted TGF-βs with AAVs, and subsequently evaluate the
advantages of AAV-based TGF-β targeting in prostate
cancer.
7.1. AAVs for siRNA Mediated Silencing of
Androgen Receptor (AR)
Although no studies have thus far attempted to target
TGF-βs with AAVs in prostate cancer, several other genes,
such as the androgen receptor, have been targeted for
downregulation in attempt to catalyse tumour destru ction.
The effectiveness of this procedure was exhibited in a
study by Sun et al. [29], where short interfering RNA
(siRNA) was packaged in an AAV and injected intratu-
mourally as well as systemically. Delivery of siRNA tar-
geting AR successfully downregulated the growth of pro-
state cancer xenografts. This has great potential for tar-
geting the members of the TGF-β superfamily—the fact
that reliable infection of the virus in to the prostate shows
exciting potential for packaging a TGF-β superfamily
member to the virus to target the TGF-β signaling cas-
cades.
7.2. ProstAtak
An intriguing form of adenoviral associated therapy that
utilises the suicide gene method is ProstAtak. Suicide
gene therapy entails specific delivery of drug metaboliz-
ing enzymes into tumour cells. In the presence of a ubiq-
uitously delivered prodrug, precise metabolism of these
prodrugs into harmful metabolites is provoked in the
cancer cells, thus selectively killing them. ProstAtak ex-
ploits targeted delivery of the herpes simplex virus thy-
midine kinase (HSV-tk) into cancer cells, in conjunction
with the prodrug valacyclovir. This vaccine is currently
under Phase III human clinical trials, with very promis-
ing preliminary results. The synergistic effects of Prost-
Atak and radiation therapy incite a stimulatory effect on
the immune system, which poises it to attack remaining
residual cancer cells. Immunotherapies such as this are
becoming increasingly popular, as intrinsic “radar” sys-
tems set up by the body itself and may be a reliable and
quick detection system which will lead to subsequent de -
struction of these “runaway” cells, regardless of where in

P. Ramarao, E. Gold / Advances in Bioscience and Biotechnology 4 (2013) 8-14 13
the body they might be. Interestingly, AAV-mediated
HSV-tk also shows potential in therapies which use gan-
cyclovir in bladder cancer [30].
7.3. GVAX
Another promising clinical trial uses granulocyte ma-
crophage colony-stimulating factor (GM-CSF) to promote
cancer cell death. GM-CSF incites a potent immunosti-
mulatory response which draws the attention of the im-
mune system to the cancer cells. This factor has been us-
ed in a variety of clinical trials for different cancers, in-
cluding bladder and prostate cancers. However, GVAX is
a whole cell vaccine, which has been transduced by a re-
combinant AAV (rAAV) vector. Although this is not a vi-
ral vaccine, it does highlight the versatility of using AAVs
to develop various therapies using their role as a gene
transfer vec tor. GVAX is currently transition ing from Phase
I to Phase II human clinical trials, with very encouraging
results published in February 2012. A fixed dose of GVAX
was conducted in con junction with a dose escalation trial
of Ipilimumab, a CTLA4-blocking antibody, for patients
with me tas ta tic cas tr at ion-re sista n t pros tate c anc er (mCR PC)
[31].
8. CONCLUDING REMARKS
It is clear that AAVs pose significant advantages and po-
tential in cancer gene therapy. Although TGF-βs have not
yet been targeted with AAVs in prostate cancer, there is
compelling evidence that this may be a promising ther-
apy for metastatic, end stage prostate cancer. Overall, the
high safety profile and ability to produce long term gene
expression make AAVs a great potential therapy for
mCRPC. At present, several AAVs have been rigorously
interrogated for use in clinical applications; AAV8 for
Haemophilia and macular degeneration, AAV1, 6 and 9
for cardiomyopathies, AAV5 for cystic fibrosis and AAV1,
5 and 8 for CNS-related diseases such as Parkinson’s and
Alzheimer’s disease.
Despite the vast array of advantages involved with the
utility of AAVs for cancer therapy, the transition of th ese
therapies from the bench to the clinic is far from smooth .
For exampl e, a ltho ugh pr e-c linica l re sults sho wed enco u-
raging potential for the use of AAVs in Haemophilia B, it
was difficult to achieve therapeutic levels of Factor IX.
Additionally, there was actually a T-cell mediated re-
sponse elicited which resulted in the destruction of the
therapeutic transduced cells [32]. This shows that although
the immune responses elicited by AAVs are weaker in
comparison to other viruses, it is sufficient to hinder ap-
propriate delivery of the therapy. A valu able po int to note
is the fact that this immune response was not observed in
mouse models [33], further highlighting the difficulties
of translating therapies into the clinic.
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